The guanine nucleotide binding protein Go is composed of alpha, beta, and gamma subunits. The carboxyl-terminal regions of Go-alpha, like those of other alpha-subunits of signal transducing proteins are presumed to be the site of their interaction with receptors. Pertussis toxin-catalyzed ADP-ribosylation of a cysteine, the fourth amino acid from the carboxyl terminus of G-alpha substrates couples them from their receptor. To define the structural requirements of Go-alpha protein for ADP-ribosylation, recombinant mutants were constructed with alterations in the terminal five amino acids of the carboxy] region and their ability to serve as pertussis toxin substrates was evaluated. The results demonstrate the basic requirement for intact three penultimate amino acid residues in the carboxyl terminal region of Go-alpha: -cys351gly352-leu353- for ADP-ribosylation. Deletion of leu353 abolished the reaction, its replacement with glycine or alanine greatly reduced it. However, a mutant with leu353 replaced by valine retained its full capacity to serve as a substrate, in line with a greater similarity of these two amino acids. Modification of the carboxyl terminal mutants that can undergo ADP-ribosylation was enhanced by beta-gamma subunits, which had no effect with the totally negative mutants. A twenty-amino acid deletion mutant locking 20 amino acids at the amino terminus of the molecule was a substrate for ADP-ribosylation, but the reaction was not affected by beta-gamma, consistent with the known function of amino terminus of G-alpha subunits in binding of beta-gamma.